IntroductionMany cases of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by an activating point mutation of JAK2 (JAK2 V617F ). It has been generally accepted that JAK2 V617F -positive cells outpace normal hematopoietic cells as a result of constitutively active growth factor signaling 1 ; however, failure of JAK2 V617F to confer a significant competitive advantage over normal hematopoiesis in 2 independent knock-in MPN models 2,3 suggests that additional factors may be required to promote expansion of JAK2 V617F -positive cells in patients.As MPN patients overproduce certain proinflammatory cytokines known to suppress normal hematopoiesis, 4 it is conceivable that JAK2 V617F may protect mutant stem cells and progenitors from the apoptotic cues induced by these cytokines.In this context, we recently observed that TNF␣ levels are elevated in mice with retrovirally induced JAK2 V617F MPN. 5 The physiologic effects of TNF␣ are complex and cell type-dependent, ranging from stimulation of proliferation to induction of apoptosis. 6 TNF␣ negatively regulates the expansion and self-renewal of pluripotent hematopoietic stem cells (HSCs) 7,8 and has inhibitory effects on normal as well as some leukemic human hematopoietic progenitor cells. [9][10][11] TNF␣'s involvement in the evolution of leukemia is not without precedent. Studies in Fanconi anemia (FA) have implicated TNF␣ hypersensitivity as a central mechanism of clonal evolution and progression to acute myeloid leukemia. In the FA Complementation Group C murine model (Fancc Ϫ/Ϫ ) TNF␣ induces bone marrow failure 12 and can promote the evolution of somatically mutated TNF␣-resistant preleukemic stem cell clones. 13 Taking into account TNF␣'s role in clonal evolution and that elevated TNF␣ levels are present in human MPN we hypothesized that JAK2 V617F induces TNF␣ expression and simultaneously confers TNF␣ resistance to MPN progenitor cells. Methods Isolation and culture of primary cellsBlood mononuclear cells (MNCs) were obtained from peripheral blood samples of patients with polycythemia vera (PV) and essential thromobocythemia (ET), myelofibrosis (MF), or normal volunteers. CD34 ϩ cells were obtained from bone marrow of normal, PV and ET patients or peripheral blood of MF patients. All patients gave their informed consent in accordance with the Declaration of Helsinki to participate in the study, which was approved by the Institutional Review Boards of Oregon Health & Science University (OHSU), Portland Veterans Affairs Medical Center, Cornell University, and Freiburg University.Submitted April 13, 2011; accepted August 7, 2011. Prepublished online as Blood First Edition paper, August 22, 2011; DOI 10.1182 DOI 10. /blood-2011 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''adverti...

SUMMARY Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.

Summary Therapeutic vaccination using T‐cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR‐specific T cells. To evaluate induction of regulatory T‐cell subtypes, immunological and clinical parameters were followed in 23 treatment‐naïve subjects with relapsing‐remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open‐label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR‐reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)‐10‐secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both ‘native’ CD4+ CD25+ and ‘inducible’ CD4+ CD25− peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.

Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.